What is PCR?
Sometimes called "molecular
photocopying," the polymerase chain reaction (PCR) is a fast and
inexpensive technique used to amplify, or make many copies of, small segments of
DNA. This is necessary because methods used for analyzing DNA
(determining the DNA base pair sequence) require more DNA than may
be in a typical sample. A particularly useful feature of PCR
is that it allows the amplification process to be limited to
specifically targeted segments of the DNA mixture--such
as the Y chromosome markers used in genealogical testing.
When your vials of cheek cells arrive
at the lab for testing, they are first mixed with a detergent which
causes the cells to burst open and release their DNA along with other
cell contents. The mixture is then washed with a phosphate
containing buffer (mild salt) solution to dilute cellular debris. With
minimal preparation, the sample is ready and the DNA on the targeted
area of a chromosome can be amplified.
How does PCR work?
PCR is a process based on the ability
of a DNA polymerase enzyme that can
synthesize a complementary strand to a targeted segment of DNA in a
test tube mixture of the
bases. In addition, the mixture must also contain two DNA
fragments, each about 20 bases long, called
primers, that have sequences complementary to areas adjacent
to each side of the target sequence. (To do PCR, you need to
know the DNA sequence around the region you want to amplify.) These
primers can be constructed in the lab, or purchased from commercial
suppliers. If chosen well, the 20-25 base pair sequence will
be unique in the entire human genome so will match only the place specifically chosen
thus limiting and defining the area to be copied. (For a more
detailed discussion of primers and PCR,
click here )
The mixture is first heated to
denature (separate) the sides of the double- stranded DNA and then cooled to allow (1) the primers to find and
bind to their complementary sequences on the separated strands and
(2) the polymerase to extend the primers into new complementary
strands. Repeated heating and cooling cycles multiply the target DNA
exponentially, since each new double strand separates to become two
templates for further synthesis. In about 1 hour, 20 PCR cycles can
amplify the target by a millionfold. In 32 cycles at 100%
efficiency, 1.07 billion copies of targeted DNA region are created.
From: The National Human Genome Research
Office of Science Education and Outreach
Also see an animation
of the PCR at
The entire cycling process of PCR is
automated and can be completed in just a few hours. It is directed
by a machine called a thermocycler, which is programmed to alter the
temperature of the reaction every few minutes to allow DNA
denaturing and synthesis. To avoid the destruction of needed
enzymes in the mixtures by the high temperatures needed to
denature the DNA, enzymes from bacteria that thrive in hot
springs are used for the process.
Why is PCR useful?
Once amplified, PCR products can be
used in many different laboratory procedures; for example, most
mapping techniques in the Human Genome Project rely on PCR.
PCR is also valuable in a number of
newly emerging laboratory and clinical techniques, including DNA
fingerprinting, detection of bacteria or viruses (particularly
AIDS), and diagnosis of genetic disorders and preparing
samples for genealogical DNA testing.
Click here to find out how STR's are counted.